Mycobacterium tuberculosis, the etiologic agent of tuberculosis (TB) in humans, continues to be a major health problem worldwide. Mycolic acids, one of the most important lipids of the outer membrane of mycobacteria, have been largely associated with bacterial virulence and antibiotic resistance and its biosynthesis pathway is one of the main targets for TB treatment. Biosynthesis of mycolic acids involves two structural distinct fatty acid synthase systems, FAS-I and FAS-II, which should work in a finely coordinate manner to keep lipid homeostasis tightly regulated. The main goal of this proposal is to understand how mycobacteria exert this exquisite control over the biosynthesis of their membrane lipids and find out the key components of the regulatory network that control fatty acid and mycolic acid biosynthesis at the transcriptional level. To achieve these goals we propose to use a multidisciplinary approach including genetic analysis of conditional mutant strains in fas and mabR (the transcriptional regulator of the main fasII operon), both in Mycobacterium smegmatis and M. tuberculosis, the identification and characterization of the metabolic signals that are sensed by MabR and any other regulatory proteins involved in the FAS-I/FAS-II regulatory network, by using biochemical and spectroscopic techniques, and finally the determination of MabR structure by X-ray crystallography. A better understanding of this complex process of regulation of lipid homeostasis in mycobacteria will greatly contribute to the development of new strategies to control this disease, including the design or identification of compounds that could deregulate fatty acid biosynthesis and induce bacterial death. PUBLIC HEALTH RELEVANCE: Since fatty acid biosynthesis is essential and energetically expensive, organisms have developed mechanisms that maintain the concentration of lipids tightly regulated. In M. tuberculosis, the etiological agent of tuberculosis, fatty acid biosynthesis is even more relevant since these molecules are also the precursors of other more complex lipids, the mycolic acids, which are essential for conferring virulence and antibiotic resistance to these organisms. Biosynthesis of these metabolic pathways needs to be tightly regulated and disruption of this regulation should be highly damaging for cell survival. Our project proposes to identify and characterize all the components of this complex net of regulation and hopefully identify new drug targets that could act by globally affecting lipid metabolism.